Ongoing New Infection?: Detection of Circular Viral DNA in PBMC of
HIV-Infected Individuals with No Detectable Viral RNA.

With the success of highly active antiretroviral regimens, a significant
number of patients have no detectable plasma viral RNA by the most sensitive
assays available. These patients however, have persistent, replication competent
virus as determined by in vitro culture as well as detectable viral DNA. To gain
a clearer understanding of the state of the virus in these patients, we examined
PBMC for the presence of closed circular forms (cc) of extrachromosomal HIV DNA.
While high levels of cc DNA are seen in acutely infected PBMC they rapidly
decline with a short half-life, therefore, detection would be suggestive of
ongoing new infection in vivo.

DNA was extracted from the PBMC isolated from 35 patients with plasma viral
loads of <50 copies HIV RNA/ml of blood. All of the patients were on
multi-drug antiretroviral regimens. The mean CD4+ cell count was 493 cells/mm3
(33-984) and the mean duration of viral suppression was 13 months (3-27.5 mths).
Low molecular weight (LMW) DNA was specifically enriched from the cell pellet
during extraction. The PCR reaction was a modification of that previously
described ( Cara A. et al, Virology 208, 242-248) with the addition of an
initial round of amplification using external primers to enhance sensitivity. A
total of 70% of the patients had detectable cc DNA using the nested
amplification while only 7% were positive using a single round of amplification.

While the presence of cc DNA in the majority of patients with no detectable
plasma RNA suggests that there is ongoing de novo infection, further evaluation
of the stability of this form of extrachromsomal DNA in other viral reservoirs
is needed. Longitudinal studies are required to determine whether the presence
of cc DNA is predictive of duration of viral suppression (or viral
breakthrough).